Cloning and functional analysis of the molting gene CYP302A1 of Daphnia sinensis

Background Molting is an important physiological process in the growth and development of arthropoda, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is a key enzyme which plays a critical role in the synthesis of ecdysone in insects, but it has not been identified in cladocera. Results The CYP302Al gene of Daphnia sinensis was cloned and its function was analyzed in this paper. The CYP302Al gene of D. sinensis was 5926 bp in full-length, with an open reading frame (ORF) of 1596 bp that encoded 531 amino acids (aa), a molecular weight of 60.82 kDa and an isoelectric point of 9.29. The amino acid sequence analysis revealed that there were five characteristic conserved regions of cytochrome P450 family (namely helix-C, helix-K, helix-I, PERF and heme-binding). In dsRNA mediated experiment, the expression level of CYP302A1 gene decreased significantly (knock-down of 56.22%) in the 5% Escherichia coli concentration treatment. In addition, the expression levels of EcR and USP and HR3 genes in the downstream were also significantly decreased, whereas that of FTZ-f1 gene increased significantly. In the 5% E. coli treatment, the molting time at maturity of D. sinensis prolonged, and the development of embryos in the incubation capsule appeared abnormal or disintegrated. The whole-mount in situ hybridization showed that the CYP302A1 gene of D. sinensis had six expression sites before RNA interference (RNAi), which located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine. However, the expression signal of the CYP302A1 gene of D. sinensis disappeared in the first antennal ganglion and obviously attenuated in the ovary after RNAi. Conclusion The CYP302A1 gene played an important role in the ecdysone synthesis pathway of D. sinensis, and the knock-down of the gene affected the molting and reproduction of D. sinensis.


Background
During the life history of cladocera (e.g. Daphnia), their growth and molting are alternately [1]. The molting action runs through their whole life cycle, and it is a necessary step before they grow and reproduce [2].
Molting is a result of long-term evolution in arthropod, which are regulated by many factors [3]. In crustacean ecdysis, ecdysteroid is the most important regulatory factor, which the expression levels vary among species [4]. Among ecdysones, 20-hydroxyecdysone (20E) is one of the more active hormones in insects [5]. Moreover, ecdysone can not only regulate the molting physiology in arthropods, but also plays important roles in their growth, reproduction and phenotypic plasticity [6][7][8][9][10].
As one of the key genes in insect ecdysone synthesis, CYP302A1 that can catalyze the carbon-22 hydroxylase is a member of the mitochondrial cytochrome P450 family [28][29][30][31]. Chavez et al. (2000) found that inactive ecdysone and 20E had lower titers in the CYP302A1 (DIB) mutant embryos of Drosophila, and two 20E-inducible genes (IMP-E1 and L1) failed to express in some tissues, resulting in anaphase abnormality in morphology [13]. After RNAi in Sogatella furcifera and Laodelphax striatellus, the expression levels of both CYP302A1 gene and ecdysone receptor (EcR) gene decreased significantly and the development and death time of nymphs delayed [31]. In spatio-temporal expression profiling of Bombyx mori, CYP302A1 gene showed a higher expression in the ovary, testis and head of the larvae [32]. A few investigations have reported on the genes related to the ecdysone synthesis pathway of Cladocera [25][26][27]33], but the molecular mechanisms of ecdysone synthesis pathway and ecdysone signal transduction pathway need still to be further revealed.
The study on molecular biology of Daphnia species has become a hot spot, with the successive reports on the genome of Daphnia pulex and D. magna [34,35]. In this study, based on the transcriptome, real-time PCR and RNAi technologies, the CYP302A1 gene cloning, and the changes of downstream response gene expressions and individual phenotypic characteristics after knock-down of the gene were analyzed in D. sinensis. Meanwhile, the function of the CYP302A1 gene was discussed. Moreover, the expression sites of the CYP302A1 gene in D. sinensis was also detected by whole mount in situ hybridization technique. Our results will help to clarify the ecdysone synthesis pathway of Daphnia species, and provide a reference for the future study of ecdysis-related signaling pathways.

Sequence and phylogenetic analysis of CYP302A1 gene
The full-length of the CYP302A1 gene in D. sinensis is 5926 bp with the open reading frame (ORF) of 1596 bp, which encodes 531 amino acids. The molecular formula of its protein is C 2743 H 4344 N 750 O 778 S 17 , with a molecular weight of 60.82 kDa and an isoelectric point of 9.29. Moreover, there is no signal peptide sequence and transmembrane domain in the CYP302A1 gene. Compared with other arthropods, the CYP302A1 gene of D. sinensis had the highest homology with Tigriopus japonicus (45.99%). In the amino acid sequence of the CYP302A1 gene, there were five characteristic conserved domains (namely, helix-C, helix-K, helix-I, PERF and heme binding) (Fig. 1). The phylogenetic tree indicated that D. sinensis was the closely related to the two other Daphnia species D. magna and D. pulex, followed by Tetranychus cinnabarinus (Fig. 2).

Induced expression of dsRNA
Usually, the expression fragments of empty body, L4440-EGFP (Enhanced Green Fluorescent Protein) recombinant plasmid and L4440-DIB recombinant plasmid induced by IPTG are 163 bp, 913 bp and 983 bp, respectively. 1% Agarose gel electrophoresis showed that the L4440 vector plasmid and L4440-EGFP were about 150 bp and 900 bp in size, respectively (Fig. 3A), and the L4440-DIB recombinant plasmid was about 1000 bp (Fig. 3B). Therefore, these results were consistent with the expected lengths of the plasmids.

mRNA expression of the CYP302A1 gene after RNAi
Compared with the control treatment, the mRNA expression levels of the CYP302A1gene of D. sinensis in the 5% and 10% E. coli treatments decreased by 68.34% and 23.32%, respectively (Figs. 4,5). Under the 5% E. coli concentration, the expression levels of EcR, USP and HR3 genes in the downstream decreased significantly, whereas the expression levels of FTZ-f1 gene increased significantly (Fig. 4). Under the 10% E. coli concentration, there was no significant difference between E10-DIB treatment and E10-EGFP treatment. Moreover, the expression levels of USP and HR3 genes in the downstream decreased whereas that of the FTZ-f1 gene increased, however, no significant differences were observed (Fig. 5). These results indicated that the dsRNA-DIB containing 5% E. coli concentration inhibited significantly the expression level of the CYP302A1 gene in D. sinensis whereas the interference efficiency was low under higher E. coli concentration (10%).

Phenotypic changes of D. sinensis after RNAi
Both no. eggs at first brood and no. offspring at first reproduction of D. sinensis in the E5-EGFP treatment were bigger than those in the E5-DIB treatment. However, no. eggs at first brood of D. sinensis at the 12th day of the experiment in the E5-EGFP treatment was significantly bigger than one in the E5-DIB treatment (Fig. 6). The molting time at first brood of D. sinensis in the E5-EGFP treatment were shorter than those in the  E5-DIB treatment as well as the molting time at first reproduction ( Fig. 6).

Positioning analysis of the CYP302A1 gene in D. sinensis
Whole mount in situ hybridization showed that the CYP302A1 gene in D. sinensis had six expression sites, which respectively located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine (Fig. 7A). After RNAi, the expression signal of the CYP302A1 gene disappeared in the first antennal ganglion of D. sinensis, and the expression signal at the ovary was also greatly attenuated (Fig. 7B). Similarly, the expression sites of the CYP302A1 gene in D. sinensis was not detected in the negative control experiment (Fig. 7C).

Discussion
Ecdysone is synthesized under the catalyzation of a series of cytochrome P450 family coding enzymes in arthropods, which can regulate basic physiological processes such as molting and reproduction in arthropods [14,29,33]. The cytochrome P450 family is an ancient gene family that exists in almost all organisms [38,39]. Although the amino acid sequences of the cytochrome P450 family member have high variability, a certain conservative domain are still found. In insects, conserved domains include helix-C, helix-I, helix-K, PERF and heme-binding [13]. In this study, the CYP302A1 gene of D. sinensis contained also the above five conserved domains, indicating that CYP302A1 gene belonged to the cytochrome P450 family.
In recent years, with the development of RNAi technology [40], RNAi has been widely used in the study of biological gene function [41,42]. Using chitin synthase gene A (SeCHSA) as the target gene, the growth and development of Spodoptera exigua larvae fed by E. coli containing dsRNA of SeCHSA was disturbed, and then the mortality rates in the 5th instar larvae increased significantly [43]. After either feeding or injecting dsRNA of the sex-determining gene Transformer-2 to Zeugodacus scutellata, the Transformer-2 gene were all silenced, and increased significantly the number of male among their offspring [44]. Through RNAi to the appendage terminal Distal-less (Dll) gene of D. magna, it was found that the appendage terminal was deficient [45]. In this study, the expression levels of the CYP302A1 gene decreased significantly (knock-down of 68.34%) in the 5% E. coli treatment, whereas it was only knock-down of 23.32% in the 10% E. coli treatment, indicating that the silencing effect at the lower E. coli concentration was better than at higher concentration. This phenomenon was also observed in other Daphnia species [46,47]. Through the feeding experiment on different concentrations of E. coli expressing the phenoloxidase dsRNA, Schumpert et al. (2015) found that the overall % with clear carapace (20%) at the end of the experiment of Daphnia melanica under higher concentration was lower than one (60%) under lower concentration [46]. After the 14-days feeding of dsRNA-Dhb2, Eytcheson and LeBlanc (2018) found also that the mRNA levels of Dhb2 of D. magna under higher E. coli concentration was significantly lower than one under lower concentration [47]. Therefore, it is likely that more bacteria consumption under higher E. coli concentration may have resulted in a decrease of dsRNA delivery and attenuation of siRNA suppression in D. sinensis. Moreover, the optimal concentration and time of silencing target genes can depend on different experimental animals or genes. After injecting dsRNAs of CYP307A2 and CYP314A1 genes, the development of the ovaries in female adults of Agasicles hygrophila delayed, and the egg production dropped significantly, and the expression level of vitellogenin gene (Vg) down-regulated significantly [48]. Similarly, when injected with dsRNA of the CYP315A1 gene, the adult ovary in Plutella xylostella became smaller and mature eggs decreased, and the cumulative number of eggs also decreased significantly [49]. In this study, the expression levels of the ecdysone receptor EcR gene and the USP gene in D. sinensis belonging to the downstream response genes of ecdysone decreased significantly after RNAi. Moreover, the knockdown of the CYP302A1 gene resulted in a significant decrease in the expression level of the downstream HR3 gene, but no significant effect on E75 gene was observed.
During the experiment, the destruction of these downstream genes could result in some aborted eggs or dead embryos of D. sinensis in the incubation capsule. Hannas and Leblanc (2010) also found that the ecdysone could significantly affect the expression level of HR3 gene but less effect on E75 gene [50]. In this study, the knock-down of the CYP302A1 gene increased significantly the expression level of the FTZ-f1 gene in D. sinensis. Usually, FTZ-f1 is mainly responsible for regulating the expression of upstream CYP302A1, CYP306A1 and CYP315A1 genes in Drosophila ecdysone signal transduction [30]. Therefore, our results strongly supported that the CYP302A1 gene is an ecdysone synthesis pathway gene in D. sinensis, affecting the molting and reproduction of Daphnia. Rewitz et al. (2006) found that the ecdysone synthesis pathway gene CYP302A1 of the tobacco hawkmoth was mainly expressed in the prethymocytes during the larval stage whereas it was detected in the fat body, midgut, ganglia, malpighian tubules and epidermis in animals after the fifth ecdysis [51]. In this study, the CYP302A1 gene in D. sinensis had mainly six expression sites, which located in the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine. However, only expression signal of the CYP302A1 gene in the first antennal ganglion of D. sinensis disappeared after RNAi. Moreover, the expression signals in the ovary weakened greatly. Usually, the first antennae are the sites of signalling for the hormonal induction of reproduction of cladocera. Therefore, the CYP302A1 gene can be involved in the reproductive transformation of D. sinensis. It was also consistent with the aborted eggs or dead embryos in the incubation capsule of D. sinensis under the 5% E. coli concentration. Sumiya et al. (2014Sumiya et al. ( , 2016 found that both Neverland1 and CYP314A1 are involved in the synthesis of ecdysone in D. magna, and intestinal epithelial cells were responsible for this synthesis [25,26]. Usually, the cecum is located in the left and right sides of the front end of the midgut in cladocera, with a pair Fig. 4 qPCR results of ecdysis gene CYP302A1 and its downstream response gene in D. sinensis fed by 5% E. coli concentration. Note: E5-EGFP: 5% E. coli concentration containing L4440-EGFP; E5-DIB: 5% E. coli concentration containing L4440-DIB. *stands for P < 0.05; **stands for P < 0.01. EcR: ecdysone receptor; USP: Ultraspiracle; E75: ecdysone-induced protein 75, which is a nuclear hormone receptor; HR3: hormone receptor 3; FTZ-f1: Fushi-tarazu factor 1, which is an ecdysone-inducible transcription factor. All these genes respond to the regulation of 20-hydroxyecdysone of ear-like appendages. In this study, the expression site of the CYP302A1 gene at the cecae of D. sinensis is consistent with other studies [25,26]. It was likely that the cecum in D. sinensis was an important synthesis and secretion site of ecdysone, and some sites of the thoracic limb and tail spine began to express the CYP302A1 gene during the ecdysis. In conclusion, the CYP302A1 gene in D. sinensis was a gene related to synthesis of the ecdysone, which would play an important role in the molting and reproduction of cladoceran.

Conclusions
Molting is an important physiological process in the life history of cladocera, which is mainly regulated by juvenile hormone and ecdysone. CYP302A1 is the key enzyme which plays a critical role in the synthesis of ecdysone of insects, but it has not been identified in cladocera. In this study, the CYP302A1 gene was also found in D. sinensis. The amino acid sequence analysis revealed that the CYP302A1 gene of D. sinensis had five characteristic conserved regions of cytochrome P450 family, namely, helix-C, helix-K, helix-I, PERF and heme-binding. In dsRNA mediated experiment, the expression level of the CYP302A1 gene decreased significantly in the 5% E. coli treatment. Meanwhile, the expression levels of EcR, USP and HR3 genes in the downstream decreased also significantly whereas that of FTZ-f1 gene increased significantly. Moreover, the development of embryos in the incubation capsule of D. sinensis appeared abnormal or disintegrated. The whole-mount in situ hybridization indicated that the CYP302A1 gene of D. sinensis had six expression sites (namely the first antennal ganglion, ovary, cecae, olfactory hair, thoracic limb and tail spine) before RNAi.

D. sinensis culture
D. sinensis were obtained from the hatching of resting eggs in the sediments of Lake Chaohu, China. The individual was monoclonally cultured in an intelligent light incubator at 25 °C, with a 12 h:12 h light/dark cycle. The culture medium was changed every day, and D. sinensis were fed with 2 × 10 5 cells/mL of Tetradesmus obliquus. The culture medium was filtered and aerated tap water over 48 h.

RNA extraction and first-strand cDNA synthesis
50 female adults of D. sinensis were collected and stored in 100 μL RNAlater (Biosharp, Hefei, China) in 1.7 mL tubes, and total RNA were extracted by the MiniBEST universal RNA kit (TaKaRa, Dalian, China). The quality and purity of RNA was measured using a NanoDrop spectrophotometer (MD2000D, Biofuture, England) and Agarose electrophoresis. The first-strand cDNA was synthesized using the PrimeScript ™ RT kit (TaKaRa) according to the manufacturer's instructions, and then stored at − 80 °C.

Sequence and phylogenetic analysis of CYP302A1 gene
The full-length CYP302A1 gene was obtained by sequencing, splicing and functional annotation of the D. sinensis transcriptome in our previous investigations [52]. The open reading frame of the nucleotide sequence of the CYP302A1 gene was analyzed using the online prediction tool ORF finder (https:// www. ncbi. nlm. nih. gov/ orffi nder), and the amino acid sequence was obtained. The amino acid deduction analysis and alignment of the CYP302A1 gene were performed by DNAMAN software, and the phylogenetic tree was constructed by MAGA 11.0 software. Isoelectric point analysis were executed using ExPASy ProtParam (https:// web. expasy. org/ protp aram). Signal peptide and transmembrane region in protein were respectively predicted using Signal 4.1 Server (http:// www. cbs. dtu. dk/ servi ces/ Signa lP-4.1) and TMHMM (http:// www. cbs. Dtu. dk/ servi ces/ TMHMM).

Induced expression of dsRNA
The primers were designed according to the transcriptome data and EGFP (Enhanced Green Fluorescent Protein) plasmid sequence (Table 1). EGFP was used as a negative control [46,53]. The PCR program was as follows: 95 °C for 3 min, 95 °C for 15 s, 55-60 °C for 15 s, and 72 °C for 40 s, followed by 35 cycles, and 72 °C for 5 min. PCR products were detected by a 1% agarose gel electrophoresis. PCR products of DIB/EGFP were subcloned into the pEASY-Blunt3 cloning vector (TransGen, Beijing, China) and sequenced (General Biol, Nanjing, China). After sequencing, the expression vectors L4440 and pEASY-Blunt3-DIB/EGFP plasmid were digested using restriction enzymes BamH I and Xho I (TaKaRa), and then ligated. The L4440 vector contains two T7 promoters which can be induced by isopropyl β-d-1thiogalactopyranoside (IPTG) to produce dsRNA of the sequence ligated between these promoters. The L4440 constructs were transformed into E. coli DH5α cells (Sangon Biotech, Shanghai, China), and the vector was confirmed by sequencing (General Biol). After sequencing, the L4440-DIB plasmid was transformed into E. coli HT115 cells (a strain deficient in RNase III and an efficient production for dsRNAs). The transformed cells were cultured overnight in LB medium containing ampicillin (100 μg/mL, Sangon Biotech) and tetracycline (12.5 μg/mL, Sangon Biotech) for the CYP302A1 RNAi experiments. Isopropyl IPTG (1.0 mM, Sangon Biotech) was added to induce the T7 RNA polymerase and subsequent production of dsRNA of the target sequence. The expression of dsRNA was detected by 1% agarose gel electrophoresis. The primers used in the experiments were listed in Table 1.

RNAi feeding protocol
Four food treatments were selected for the experiments, namely, E5-EGFP: 5% dsRNA-EGFP + 95% T. obliquus; E5-DIB: 5% dsRNA-DIB + 95% T. obliquus; E10-EGFP: 10% dsRNA-EGFP + 90% T. obliquus; E10-DIB: 10% dsRNA-DIB + 90% T. obliquus. There were three replicates at each food treatment. Total food biomass was 20 mg/L wet weight. 15 animals (birth time < 12 h) in each replicate were employed as the mother. During the experiment, the culture medium at each food treatment was refreshed every day, and all newborns produced by the mother were immediately removed. All mothers in each replicate were collected at the twelfth day after feeding, placed in a 1.7 mL tube containing 100 μL RNAlater and stored in a refrigerator at 4℃ for Table 1 Names and sequences of primers used in the experiment DIB and EGFP are interference primer, with length of the amplicons is respectively 820 bp and 750 bp. The other primers are qPCR primer, and the underlines of forward and reverse primer are the restriction endonuclease sequence 12 h, and then transferred to an ultra-low temperature refrigerator at − 80℃. After RNAi, the expression levels of related genes were determined by qPCR, and the relative expression levels of target genes was calculated by 2 −△△ Ct. The life history parameters of the four growth stages (at birth, at first brood, at first reproduction and at the twelfth days) were observed and recorded during the experiment.

Whole mount in situ hybridization
In order to prepare probes for in situ hybridization according to the ORF of the CYP302A1 gene, the sequences of specific primers were designed as follows: ISH-DIB-Forward: CGC GGA TCC GAG CTT TAT ACT GTA TCA TCT TGCC, ISH-DIB-Reverse: CCG CTC GAG GAC TCT TTT ACT GCA GCC TTT AGAT, which the length of the amplicon is 150 bp. Target fragment was synthesized according to the primer sequence. After sequencing, the positive clone bacteria were amplified and cultured, and then the Blunt3-ISH-DIB vector plasmid was extracted. The concentration and purity of the plasmid were determined by a NanoDrop spectrophotometer (MD2000D, Biofuture). The linearized plasmid was obtained through restricted digestion of BamH I or Xho I, and the digested DNA fragments were purified and used as templates for sense and antisense probes, respectively. RNA probes were synthesized through DIG RNA Labeling Kit (SP6/SP7) (Roche, Mannheim, Germany), and then digested the probe cDNA template using DNase (RNase-free). Probe synthesis system (20 μL): linear plasmid ≤ 1 μg, 10 × NTP labeling mixture 2 μL, 10 × transcription buffer 1 μL, RNase Inhebitor 1 μL, RNA Polymerase SP6/T7 2 μL, and then added to 20 μL with RNase-free H 2 O. In addition, a 1/9 volume of 5 M LiCl and 2 volumes of absolute ethanol were added, and were incubated overnight at − 20 °C. RNA pellets was washed twice with 75% ethanol, and then dried to remove residual ethanol. Finally, RNA pellets was re-suspended in 30 μL diethylpyrocarbonate water, which 1 μL RNA inhibitor (20 U) were added. Aliquots of RNA solutions (1 µL) were added and electrophoresed, and the concentrations were measured. Remaining RNA probes were stored at − 20 °C.
According to the RNAi feeding protocol, 50 female adults of D. sinensis were collected. All samples were fixed in 4% paraformaldehyde (PFA) overnight, and then were replaced by anhydrous methanol and remained at − 20 °C. Whole-mount in situ hybridization was carried out according to previously published methods [36,37] with some modifications. The specimens stored at − 20 °C were rehydrated gradually with methanol-PBST and digested with proteinase K (10 µg/mL, Solarbio, Beijing, China). The individuals were digested at 37 °C for 12 min. After pre-hybridization at 68 °C for 2.5 h, 100 µL RNA probe which was diluted 1:100 was added and incubated at 70 °C overnight. The specimens were blocked for approximately 2 h at room temperature with slow shaking in MAB block solution, and then added anti-DIG antibody (diluted 1:5000, Roche) and incubated at 4 °C for 13 h. Finally, antibody solution was discarded and the specimens were washed in MABT. At room temperature, the NBT liquid dye (Roche) was used to shade the color for 15 min − 2h, and then the individuals were fixed in 4% PFA for 20 min. Hybridization was observed with a fluorescence microscope (Olympus, CX21).